Hesperetin attenuates LPS‐induced the inflammatory response and apoptosis of H9c2 by activating the AMPK/P53 signaling pathway

Abstract Introduction Hesperetin (HES), whose main pharmacological effects are anti‑inflammatory and cardioprotective properties. In our study, we investigated the role of HES in lipopolysaccharide (LPS)‐induced inflammation and apoptosis in H9c2 cells. Methods Cell viability was assessed through MTT assay. Tumor necrosis factor (TNF)‐α and interleukin (IL)‐β expression were quantified through RT‐qPCR assay. Secondly, the apoptosis rate was assessed by Terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling assay. Finally, B‐cell lymphoma 2 (Bcl‐2)‐ associated X protein (Bax), adenosine monophosphate‐activated protein kinase (AMPK), and P53 expression were quantified through western blot assay. Results Our results demonstrated that LPS stimulation decreased the cell viability, increased IL‐1β and TNF‐α expression in H9c2 cells. However, HES treatment significantly increased the cell viability, decreased IL‐1β and TNF‐α expression in LPS‐induced H9c2 cells. In addition, HES significantly increased the phosphorylation level of AMPK. Meanwhile, HES prevented against LPS‐mediated the P53 and Bax protein upregulation, and Bcl‐2 protein downregulation in H9c2 cells. More interestingly, compound C (an AMPK inhibitor) treatment eliminated the protective effects of HES. Conclusion Our findings revealed that HES attenuated the LPS‐mediated inflammation and apoptosis of H9c2 cells by activating the AMPK/P53 signaling pathway, suggesting that HES may be a potential cardioprotective agent.


| INTRODUCTION
The pathogenesis of cardiovascular disease is complex, and myocardial disease is one of the diseases with high incidence in cardiovascular disease. 1,2 Accumulating evidence demonstrates that cardiomyocytes' apoptosis and inflammatory responses are key processes in cardiomyopathy. Moreover, inflammation and apoptosis of cardiomyocytes are also prominent pathological features in other cardiovascular diseases such as sepsis, nerve damage, and myocardial infarction injury. [3][4][5][6] While traditional therapies to prevent cardiomyocyte inflammation and apoptosis remain ineffective, research has focused on new strategies. [7][8][9][10][11] Lipopolysaccharide (LPS) is involved in regulating the initiation of pathophysiological cascades. 12 Previous literature reported that reduction of LPS could improve outcomes in patients with cardiac disease. 13 LPS treatment of H9c2 cells can lead to an inflammatory response. 14 H9c2 cardiomyocytes have also been used to construct disease models, providing a new scientific approach to the treatment of chronic heart failure.
Hesperetin (HES) is an active ingredient from flowers, with pharmacological activities such as anti-inflammatory, neuroprotective, and cardioprotective. [15][16][17] In addition, HES suppresses inflammation and protects prostate endothelial cells in animal models. 18,19 Unfortunately, the exact mechanism of LPS-induced apoptosis and inflammatory response in H9c2 cells remains unclear.
Adenosine monophosphate-activated protein kinase (AMPK) as a regulatory protein was involved in cellular energy metabolism and cell apoptosis. [20][21][22] Activation of AMPK leads to the accumulation of protein p53, which induces cardiomyocyte apoptosis. 23 Our results revealed that HES attenuated the H9c2 cells' inflammation response and apoptosis induced by LPS by promoting the activation of the AMPK/P53 signaling.

| Ethical statement
The study was approved and conducted under the ethical committee of Yuhuangding Hospital. This study was approved by the clinical research ethics committee of Yuhuangding Hospital.

| Cell treatments
First, HES (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO), then HES with different concentrations (5,10,15,20, and 40 µM) was dissolved in the medium at the concentration of LPS (1 µg/mL), and the cells were cultured for 24 h.

| AMPK inhibitor
H9c2 cells were exposed to compound C (an AMPK inhibitor, 10 µM), for 30 min before adding HES. Incubation for 24 h, followed by LPS treatment of cells for 24 h. In this way, compound C treatment on LPSinduced H9c2 cells was evaluated.

| MTT assay
H9c2 cells (5 × 10 3 cells/well) were cultured in 96-well plates. After incubation with compound C (10 µM) for 1 h and/or HES for 24 h, these cells were then treated with LPS for 24 h. Then, 10 µL MTT solution (5 mg/mL, Sigma-Aldrich) was added and the plate was incubated at 37°C for an additional 4 h. After centrifugation, the cell culture medium was replaced with DMSO (Sigma-Aldrich). Finally, the absorbance value in each well of the plate was detected at 570 nm using a plate reader (Bio-Rad Laboratories, Inc.).

| Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay
Apoptosis assay was carried out by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) Apoptosis Detection Kit (Green FITC-labeled Fluorescence Assay, Universal), according to the manufacturer's protocol (Cat: KGA7072, KeyGEN BioTECH). Cells were first allowed to slide, then cells on coverslips were fixed with 4% paraformaldehyde, stained according to TUNEL kit protocol.

| Statistical analysis
Values are expressed as the mean ± standard deviation (SD). The comparison between the two groups was analyzed through the Student's t test. One-way analysis of variance (ANOVA), with Fisher's least significant difference t test, used for comparison between multiple groups. p < .05 was considered to have a significant difference.

| HES alleviated cell viability in LPS-induced H9c2 cells
H9c2 cells were first exposed to different concentrations (5,10,15,20, and 40 µM) of HES for 24 h, and then we tested the effect of HES on cell viability through MTT experiments. Compared with the control group, there was no significant difference in the survival ability of H9c2 cells when exposed to different concentrations of HES ( Figure 1A; p > .05). Our results reveal that different concentrations of HES have no significant effect on the viability of H9C2 cells. As shown in Figure 1B, LPS could significantly inhibit H9c2 cells viability compared with the control group (p < .001). However, the inhibitory effect of LPS on viability of H9c2 cells was significantly increased by HES in dose-dependent manner (p < .001).

| HES inhibited LPS-induced proinflammatory cytokine production in H9c2 cells
In Figure 2, 24 h after LPS stimulation of H9c2 cardiomyocytes, IL-1β (Figure 2A, p < .001) and TNF-α ( Figure 2B, p < .001) mRNA expression were significantly increased compared with the control group. Moreover, different concentrations of HES (5,10,15,20, and 40 µM) were exposed to assess its effect on LPSinduced IL-1β and TNF-α expression. The results demonstrated that HES treatment could significantly attenuate the LPS-induced elevation of pro-inflammatory cytokine production compared with the LPS group.

| HES attenuated LPS-induced apoptosis in H9c2 cells
As shown in Figure 3A, 48 h after LPS stimulation of H9c2 cardiomyocytes, the percentage of TUNEL-positive nuclei was markedly increased compared with the control cells (p < .001). However, HES treatment remarkably reduced the percentage of TUNEL-positive cells (p < .001 vs. the LPS-only group).
At the same time, we also detected the apoptosisrelated genes protein expression ( Figure 3B), such as Bax and Bcl-2. The result demonstrated that LPS treatment markedly increased the Bax protein expression (p < .05 vs. the control), while obviously decreased the Bcl-2 expression (p < .05 vs. the control). However, HES treatment significantly reversed the Bax (p < .05 vs. the LPS-treated cells) and Bcl-2 protein level (p < .05 vs. the LPS-treated cells). Overall, these results revealed that the HES against LPS-induced injury may be related to the apoptosis gene expression.

| HES upregulates the phosphorylation of AMPK and downregulates the P53 expression in LPS-induced H9c2 cells
To further explore the protective mechanism of HES against apoptosis, combined with previous literature, we detected the MAPK/P53 signaling pathway-related proteins in H9c2 cardiomyocytes treated with LPS. As shown in Figure 4, LPS treatment significantly decreased the phosphorylation of AMPK and increased P53 expression in H9c2 cells. However, HES treatment could reverse this effect in LPS-induced H9c2 cells. Overall, HES activates the phosphorylation of AMPK and inhibits the P53 expression in LPS-induced H9c2 cells.

| Compound C inhibits the LPS-mediated cardioprotective effect of HES
To investigate the change of the AMPK/P53 pathway in HES protection, H9c2 cells were pretreated with compound C (an AMPK inhibitor), followed by stimulation with LPS and HES. As shown in Figure 5, compound C remarkably weakened the inhibitory effect of HES on the LPS-induced reduction of AMPK phosphorylation and elevation of P53 expression. Overall, our findings revealed that compound C inhibits the LPS-mediated cardioprotective effect of HES.

| DISCUSSION
In our study, LPS treatment resulted in inflammatory and apoptotic responses in H9c2 cardiomyocytes, whereas HES markedly attenuated the inflammatory responses in H9c2 cardiomyocytes by inhibiting the proinflammatory cytokines IL-1β and TNF-α expression. Moreover, HES has a protective effect on LPS-induced H9c2 cardiomyocyte apoptosis. Finally, HES inhibits the injury response of cardiomyocytes by regulating the AMPK/P53 signaling pathway.
Myocardial inflammation has been reported in many previous studies. [24][25][26] In addition, reductions in cardiac inflammation may have beneficial effects on cardiac dysfunction. 27 LPS-induced cardiomyocyte inflammatory responses are characterized by inflammatory mediators. 28 In this study, HES treatment downregulated LPS-induced inflammatory factor expression, suggesting that HES may protect myocardial cells from inflammatory reaction to some extent.
A previous study has reported that the number of inflammatory responses and apoptosis coexisted in cardiomyocytes in the LPS-treated mouse model of sepsis. 29 Meanwhile, whether cells undergo apoptosis depends on the balance between the expression of common proapoptotic proteins and antiapoptotic proteins. 30,31 Our results reveal that HES can counteract LPS-induced cell apoptosis by inhibiting the expression of proapoptotic protein Bax and increasing the expression of apoptotic Bcl-2.
Previous studies have reported that AMPK can inhibit the proliferation of nonmalignant cells. 32 Meanwhile, P53 serves as an important regulatory factor related to cell cycle arrest and apoptosis in tumor cells. 33 Moreover, the AMPK/P53 pathway has also been proven to play a crucial role in DOX-induced cardiomyocyte death. 34,35 Therefore, in our study, in H9c2 cells treated with LPS, the phosphorylation level of AMPK protein was significantly downregulated, and the expression of P53 protein was significantly increased, while HES treatment reversed the expression of these proteins. In addition, the expression of the proapoptotic gene Bax was significantly increased in H9c2 cells treated with LPS, while the expression of Bcl-2 was significantly reduced. In conclusion, the above results confirm that the AMPK/P53 pathway is involved in LPS-induced cardiomyocyte apoptosis.
To further investigate the function of HES in cardioprotection, the effect of LPS treatment on the AMPK/P53 pathway in H9c2 cells was examined. These results showed that HES obviously attenuated the LPSinduced increase in AMPK phosphorylation level and P53 expression. In addition, AMPK, as a sensor and regulator of cellular energy, can regulate cell apoptosis and cell proliferative capacity to maintain cellular homeostasis. Therefore, our study confirmed that HES attenuated LPS-induced apoptosis of H9c2 cardiomyocytes, thereby activating the expression of AMPK and p53 proteins. Meanwhile, the AMPK phosphorylation inhibitor compound C was applied to inhibit the phosphorylation level of AMPK, and the results showed that the effects of HES on H9c2 cell apoptosis, AMPK phosphorylation, and P53 expression were reversed. Taken together, these results suggest that HES prevents LPSinduced cardiomyocyte injury by activating the AMPK/ P53 signaling pathway.
In conclusion, our study revealed that HES could protect H9c2 cardiomyocytes from LPS-induced inflammation and apoptosis by activating the AMPK/P53 signaling pathway, which provides a certain scientific basis for the prevention and treatment of LPS-induced myocardial injury.
There are also some limitations to our research, for example, (1) the main components of HES that play a role in myocarditis are not clear. F I G U R E 5 Compound C inhibits the LPS-mediated cardioprotective effect of HES. (A) AMPK phosphorylation and (B) P53 expression were analyzed by immunoblotting. ***p < .001 versus Control; ### p < .001 versus LPS + HES; & p < .05 versus LPS + HES. N = 3; data were analyzed by one-way ANOVA. The pairwise comparison after one-way ANOVA analysis was analyzed by LSD-t. ANOVA, analysis of variance; HES, hesperetin; LPS, lipopolysaccharide; LSD-t, least significant difference t test. ***p < .001 versus Control cells; ### p < .001 versus LPS; analysis; funding acquisition; project administration; supervision; validation; writing-original draft; writingreview and editing.